Tom Perkins and his group are developing three innovative nanotechnologies in their quest to understand the physics of single biomolecules. The new tools, including a stabilized optical microscope, gold nanoparticles, and RNA-based biosensors, are helping the researchers investigate nucleic acids, DNA-binding proteins, and molecular motors. The group recently developed a high-resolution optical microscope that uses stabilized laser beams to improve pointing stability and a differential back-focal plane detection method to reduce instrument noise. With specially designed calibration routines and software, the new microscope is capable of measuring subnanometer motion in three dimensions in biological systems. Using their new microscope, the group can monitor a helicase enzyme "motoring" one base at a time (approx. 0.4 nm) along a strand of DNA as it unzips the double helix. The microscope can also detect the unfolding of RNA structures in real time.

Temperature gradient around a gold bead.

In related work, the group has evaluated gold nanoparticles as a biophysics research tool. The researchers found that when gold nanoparticles are used to tether biological molecules, they significantly boost the trapping efficiency and detection sensitivity of optical-trapping assays. Because the particles are small, they make room for larger molecules such as DNA within what is often a limited detection range. The group also found that gold is susceptible to significant heating from infrared laser light (1064 nm). This means small gold beads could be used as local molecular heaters to vary the temperature for single-enzyme assays, which has been very difficult to do until now. Because gold bead temperatures can be increased tens of degrees Celsius in a few nanoseconds, the researchers can also jump the temperature of a single RNA or protein molecule and watch it thermally unfold.

The Perkins group has just begun a project to develop RNA-based biosensors in collaboration with Rob Batey, a CU biochemist. This research will capitalize on the fact that RNA molecules can be selected to bind a wide variety of specific substrates. In tackling this very challenging project, the group will harness the broad experience in RNA sciences in the University of Colorado's Departments of Chemistry & Biochemistry and Molecular, Cellular, and Developmental Biology. The researchers will couple this experience with advances in nanofluidics technology. Nanofluidics are nanometer-scale liquid handling systems that permit rapid mixing of biological samples, with observation times of seconds or longer.

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